1. Field of the Invention
This invention relates to a process for the production of an immunoglobulin G preparation having low anticomplementary activity and suitable for use in intravenous injection.
2. Description of the Prior Art
Immunoglobulin G (IgG) preparations isolated from human blood plasma are effective in the treatment of various severe infectious diseases and immunological deficiency syndrome such as hypogammaglobulinemia and agammaglobulinemia. The widely used method for isolating IgG from human blood plasma is Cohn's plasma protein fractionation method.
In Cohn's plasma protein fractionation method, IgG is obtained from Cohn fraction II or II+III. However, the recovered IgG may contain a significant amount of aggregates because IgG tends to aggregate spontaneously during storage of human blood plasma or tends to aggregate in contact with alcohol or other chemicals during the fractionation procedure, or also tends to aggregate during lyophilization. If IgG containing aggregates is administered intravenously, the aggregates may exert an anticomplement effect to cause serious anaphylactoid reactions such as hypotension, chill and fever.
Accordingly, IgG preparations for use in intravenous injection require that no aggregates are detectable when they are analyzed by the gel filtration method using a suitable carrier and that their anticomplementary activity is not greater than 20 units when determined at an IgG concentration of 5%. The term "aggregate" as used herein means any cluster formed by the combination of a plurality of IgG molecules, but does not include the dimer of IgG. It is desirable that, in addition to meeting the above requirement, the anticomplement activity is as low as possible. Thus, in order to obtain an IgG preparation for use in intravenous injection, it is necessary to remove aggregates from IgG isolated from Cohn fraction II or II+III. The methods which can be used for this purpose include:
(1) the polyethylene glycol precipitation method (i.e., the method in which polyethylene glycol is added to an aqueous IgG solution containing aggregates and the resulting precipitate of aggregates is separated by filtration); PA1 (2) the method of dissociating aggregates by reducing the pH of an aggregate-containing aqueous IgG solution to a low value such as 4 (i.e., the method in which an aqueous IgG solution is adjusted to a pH of about 4 by the addition of an acid, allowed to stand for a certain time so as to dissociate the aggregates, and then neutralized); PA1 (3) A combination of the method described in paragraph (2) above and the method of decomposing aggregates by means of a slight amount of a proteolytic enzyme (i.e., the method in which an aqueous solution of IgG is adjusted to pH about 4 and a slight amount of a proteolytic enzyme such as pepsin is added thereto so as to effect both the dissociation of aggregates by the acid and the decomposition of aggregates by the enzyme, followed by neutralization of the aqueous solution and removal of the enzyme by adsorption to an ion exchange resin); PA1 (4) the method of removing aggregates by adsorption to an ion exchange resin; PA1 (5) the method of removing aggregates by adsorption to an adsorbent such as tricalcium phosphate, activated charcoal, aluminum hydroxide or bentonite (i.e., the method in which such an adsorbent is added to an aqueous solution of IgG so as to cause aggregates to be adsorbed thereto, and the adsorbent is then separated by filtration); PA1 (6) the gel filtration method (i.e., the method in which aggregates are separated and removed by gel chromatography using a gel filtering medium, such as Sephadex G-200, having a fractionating molecular weight capable of separating aggregates from IgG monomer); PA1 (7) the membrane separation method; PA1 and the like.
As an example of the membrane separation method, there has been proposed a filtration method using a porous polymethyl methacrylate membrane which permits the passage of IgG monomer and dimer but blocks the passage of aggregates (Japanese Patent Laid-Open No. 69732/'86). Moreover, it is also known to remove aggregates by filtration through a polycarbonate membrane filter having a pore diameter of 0.05 to 0.2 .mu.m (Japanese Patent LaidOpen No. 167518/'83).
Generally, IgG (monomer) has a size of about 10 nm, IgG dimer usually contained in an aqueous solution of IgG has a size of about 12 nm, and aggregates predominantly have a size of about 14 nm and more. Thus, they are similar in size. For this reason, the abovedescribed polyethylene glycol precipitation method, the adsorption method (using an ion exchange resin, activated charcoal, tricalcium phosphate or the like) and the gel filtration method have been disadvantageous in that, even if aggregates are removed to such an extent that they are not directly detectable by gel filtration analysis, the anticomplementary activity believed to be attributable mainly to aggregates cannot be reduced satisfactorily. This means that the aggregates have not been removed to a full extent. In contrast, the membrane separation method makes it possible to remove aggregates completely and thereby reduce the anticomplementary activity satisfactorily. However, since the size of IgG monomer and dimer (hereinafter referred to collectively as IgG) is close to that of aggregates, the membrane separation method also has the disadvantage that, if it is tried to remove aggregates completely, the recovery of IgG and the treating efficiency become too low to be practicable. On the other hand, if a membrane having a relatively. large pore size is used to enhance the treating efficiency, it is difficult to remove aggregates to such an extent that the anticomplementary activity is reduced satisfactorily. Moreover, the membrane separation method also has the disadvantage that IgG tends to aggregate again at the interface between the membrane and the solution and, therefore, the resulting filtrate has a high anticomplementary activity.
Thus, it has been difficult to efficiently remove aggregates from an aqueous solution of IgG according to any one of conventionally known methods and thereby reduce its anticomplement activity to less than 10 units (at an IgG concentration of 5%). Accordingly, it would be desirable to develop a method which can remove aggregates to such a degree that they are not directly detectable and thereby produce an aqueous solution of IgG having a greatly reduced anticomplementary activity and capable of being adjusted to any desired concentration.